Normal cell development requires the regulation of transcription initiation and activation in order to express appropriate genes at appropriate times. We study regulation of transcription initiation using a simple prokaryotic system: E. coli RNA polymerase, a five subunit complex composed of a core (2 alphas, beta, and beta') and a sigma specificity factor. The sigma70 factor specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma70 is present within polymerase, two of its domains, 2.4 (an internal region) and 4.2 (the C-terminal region), interact with sequences within the -10 and -35 regions, respectively, of promoter DNA. However, in free sigma70, DNA binding is prevented by domain 1.1, the N-terminal region of the protein. To investigate the effect of domain 1.1 on the DNA binding properties of sigma70 when it is present in polymerase, we used various promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays and in assays assessing the formation and decay of stable, pretranscription complexes. We found that depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes. However, domain 1.1 does not affect the stability of these complexes once they are formed. For polymerase containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma70 recognition sequences. However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made. Our work is the first to demonstrate that domain 1.1 has a regulatory role when sigma70 is present in polymerase. We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences. The bacteriophage T4 proteins MotA, a transcriptional activator, and AsiA, a co-activator, interact with the sigma70 subunit of RNA polymerase and switch its promoter specificity from host promoters to T4 middle promoters. Middle promoters contain the sigma70 recognition sequences at -10 but lack the canonical -35 sequences. Instead they contain a 9 bp motif (a MotA box) which is centered at -30. MotA binds the MotA box as well as interacts with sigma70. We have found that the N-terminal half of MotA (MotA NTD), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E. coli 2-hybrid assay. Either replacement of the C-terminal 17 residues of sigma70 with comparable residues from another sigma, sigma38, or the sigma70 mutation R608C, located near the end of sigma70's 613 amino acids, is defective in this assay. These results suggest that the far C-terminal region of sigma70 is important for the MotA/sigma70 interaction. We have also found that a proteolyzed fragment of MotA that contains the C-terminal half (MotA CTD) binds DNA with an apparent dissociation constant that is similar to that of full length MotA. Our results support a model in which the interaction between MotA NTD and the far C-terminal region of sigma70 and between AsiA and sigma70 serve to disrupt sigma70 contacts with the -35 region of the DNA and to facilitate the binding of MotA CTD to the -30 MotA box motif.